Adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining.

OBJECTIVE: To examine the relative adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining. METHODS: Oral epithelial cells were collected from 10 healthy adults (five male, five female) and counted. Equal volumes of oral epithelial cells and candida were mixed and incubated. The epithelial cells from this mix were collected by filtration through 10 microns polycarbonate membrane filters. Cells retained on the membrane filters were stained with crystal violet followed by Papanicolaou stain. The number of yeast attached to each of 100 red, orange, and green staining oral epithelial cells was determined by direct microscopic examination. RESULTS: C albicans had a higher level of adherence (p < 0.001) to red staining oral epithelial cells (mean (SD) number of candida attached to 100 oral epithelial cells 562 (159)) than to cells staining either orange (105 (47)) or green (161 (66)). CONCLUSIONS: Oral epithelial cell variability for candidal adherence is confirmed. The technique provides an opportunity to examine the relation between oral epithelial cell type and oral candidosis in specific groups, such as tobacco smokers, where increased epithelial cell keratinisation and candidal colonisation has been reported.

p53, PCNA and Ki-67 expression in oral squamous cell carcinomas: the vagaries of fixation and microwave enhancement of immunocytochemistry.

Proliferation markers are widely used as indicators of tumour progression and aggression. Fixation and antigen retrieval methods may enhance the immunocytochemical sensitivity of these markers but may also lead to loss of specificity. As these methods are often used quantitatively, standardisation of internal and external methodology is paramount. This study aimed to compare the effects of alcohol and formalin fixation and of microwaving on the immunocytochemical demonstration of p53, PCNA and Ki-67 in oral squamous cell carcinoma using duplicate tissue blocks from 24 cases. Both qualitative and quantitative differences in antigen expression were revealed. Whilst alcohol fixation alone at least maintained and usually increased the strength of positive staining, microwaving alcohol-fixed sections often gave rise to non-specific staining. p53 staining following microwave enhancement of alcohol-fixed tissue showed a significant incidence of conversion of negative results to positive and of positive staining in unexpected tissue components. Alcohol fixation increased the sensitivity of PCNA detection with a far less dramatic loss of specificity. The results emphasise the need for careful standardisation of immunocytochemical methods, particularly when used quantitatively and for inter-laboratory comparisons.

Effects of processing at 45 C on staining.

Tissue processed at a constant temperature of 45 C including the use of paraffin wax with a melting point of 45 C displays staining characteristics that are sometimes reversed from those associated with the more usual processing schedules and wax with a melting point of 58-60 C. Staining with acid dyes, particularly in trichrome methods, are most susceptible to these changes. We suggest that this is directly related to dye molecular size and to differences in the tissue structure resulting from the heat to which the tissues were exposed.

Immunocytochemical detection of Candida albicans in formalin fixed, paraffin embedded material.

AIM: To assess the ability of the commercially available monoclonal antibody 1B12 (BioGenex, San Ramon, USA) to identify C albicans in formalin fixed, paraffin wax embedded material (FFPE). METHODS: Broth cultures of 20 strains of seven Candida species were resuspended in 4% agarose blocks, fixed in formalin for 24 hours, and embedded in paraffin wax. In addition, 16 blocks of FFPE tissue known to contain periodic acid-Schiff positive fungal hyphae were examined. Antigen retrieval involved microwave treatment of specimens in citrate buffer (0.01 M; pH 6.5) before addition of 1B12 antibody for 24 hours. Bound antibody was subsequently detected using a biotinylated link antibody and a peroxidase conjugated streptavidin. RESULTS: Only C albicans strains were 1B12 positive in the agarose blocks. All FFPE tissue blocks were found to contain 1B12 positive hyphal structures, indicating the presence of C albicans. CONCLUSIONS: The ability to identify candida organisms penetrating the lesional tissue in cases of chronic hyperplastic candidosis will help to clarify the role of individual Candida spp in this important form of oral candidosis.

Measuring infiltration during paraffin wax processing for histology.

A method is described to allow monitoring of the penetration of processing fluids into tissue during histological processing. The method is established by evaluating the effect of incorporating dimethyl sulphoxide into paraffin wax and comparing processing times with those for pure paraffin wax.

Picro-thionin (Schmorl) staining of bone and other hard tissues.

A much simplified method for the demonstration of bone morphology using Schmorl's picro-thionin technique is described, and the necessity for reliable samples of thionin emphasised. Findings from previous experiments provide the evidence and theoretical background to justify the use of certified stains for this purpose.

Lymph node micro-metastasis: an investigation, including three-dimensional reconstruction.

The mechanism by which malignant cells metastasis to, and colonise, regional lymph nodes is not known. In an attempt to characterise micro-deposits an affected lymph node associated with a primary intra-oral squamous cell carcinoma was studied. Three-dimensional reconstructions of the node and micro-tumour were made and the relationships and distribution of apparently isolated malignant cells present within the node studied. Semi-serial ethanol-fixed paraffin was sections of the node were stained with monoclonal antibody AE3 (cytokeratin). Outlines of the node and occupying micro-tumour were traced onto polystyrene tiles and onto acetate sheets, permitting the construction of both a solid and a transparent three-dimensional model. Additionally, the positions of apparently 'isolated' AE3-positive malignant cells were noted. The solid reconstruction showed that the micro-tumour deposit grew most quickly into the node via the septa and more slowly around the subcapsular region. The transparent reconstruction was less useful. Most isolated, individual malignant cells were found in the peripheral margin of the node.

Foreign body reactions and an associated histological artefact due to bone wax.

Bone wax has been used since the turn of the century as a mechanical aid to haemostasis following surgical procedures. That it may produce a foreign body giant cell reaction in a significant proportion of cases is well known. The occurrence of this material in specimens received in the laboratory is poorly documented. Four cases are described to bring attention to this exogenous agent which, it is suggested, should feature in tables of pigments and other materials occurring in tissue specimens.

Nucleolar organiser regions in odontogenic cysts and ameloblastomas.

Silver nucleolar organiser region (AgNOR) counts were performed on apical periodontal cysts, dentigerous cysts, odontogenic keratocysts, ameloblastomas and basal cell carcinomas. Significant differences, but with excessive overlap, were shown between dentigerous cysts and apical periodontal cysts and between odontogenic keratocysts and apical cysts. The mean AgNOR counts for all odontogenic cysts ranged between 2.02 and 2.65, and for ameloblastomas were 2.24, indicating that the method has neither a diagnostic nor a prognostic value in these lesions. Control oral squamous cell carcinoma tissues had significantly higher AgNOR counts than any other lesion tested.

Measuring the forces acting during microtomy by the use of load cells.

The forces acting upon the cutting blade during microtomy can be accurately measured by using a load cell. From the information obtained, the optimal knife angles (rake, clearance and slant) can be determined. In addition, the speed of cutting and thickness of sections can also be optimized. The information obtained from the load cell additionally reveals variations in tissue composition which affect the cutting forces. This paper is a preliminary communication to illustrate the possible roles for a modified microtome in (a) the study of microtomy and (b) comparative studies of tissue density.

Use of benzidine for histological demonstration of haemoglobin in human bite marks.

Macroscopic evidence of bruising from human bite marks may be inconclusive and routine histochemical methods of showing extravasated erythrocytes can be unreliable. Leuco patent blue staining, for the presence of peroxidase, Amido black B, a tinctorial staining method for haemoglobin, Perls's reaction for ferric iron (haemosiderin), Masson-Fontana for melanin, Masson's trichrome, a connective tissue strain, and the benzidine reaction for haemoglobin peroxidase were carried out in three forensic cases and one experimental case. A modified benzidine method was the most reliable indicator of haemoglobin activity, especially where dispersion into extra-cellular tissues had occurred. The resilience of the erythrocyte peroxidase enzyme to temperature changes and fixation supports the concept of a "pseudo-peroxidase" in those cells. It is concluded that free haemoglobin from bite marks, or indeed other forms of blunt trauma, may best be shown by the benzidine reaction and that exemption certificates for use of this prohibited substance may be worth pursuing.

Production of high quality ground sections of bone containing metal implants to demonstrate osseo-integration: a simplified method.

A simple method is described for examining bone-titanium implant relationship using a modified embedding technique. The method involves complete dehydration with acetone prior to infiltrating and embedding in methyl methacrylate supplemented with dibutylphthalate. Slow polymerisation is initiated with benzoyl peroxide, allowed to progress at room temperature and completed at 56 degrees C. Using this method the apparent complete apposition of bone to the titanium fixture surface has been successfully demonstrated.

Solvent systems for thin layer chromatography of biological dyes.

Thirty-nine dyes were subjected to thin layer chromatography to evaluate eight different solvent systems. The results obtained were analysed by dye class following assessment in daylight and ultra violet light. N-butanol: acetic acid: water (40:10:50) gave optimum separations of dyes viewed in visible light. Although n-butanol: ethanol: water (90:10:10) gave slightly better results for fluorescing compounds, material was frequently left at the start point with this solvent.

The effects of various fixatives on subsequent lectin binding to tissue sections.

The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin-saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate-paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin-saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.